Cardiovascular diseases represent the first cause of mortality in our modem societies. Troubles in the conduction of the electrical activity through the heart are frequently observed in these pathologies and they could lead to arrhythmias underlying the direct cause of death (Zipes and Wellens, 1998). The Purkinje conduction system is also in involved in ventricular fibrillation representing the main mechanism of cardiac sudden death in human (Haissaguerre et al, 2002). Multiples causes such as genetic and environmental factors have been advanced to explain the high incidence of arrhythmias and several genes responsible for familial diseases have already been discovered (review, (Roberts and Bragada, 2003).
Pumping function of the heart depends on the well co-ordination of cardiac contractions that are triggered by a depolarizing electrical activity. The cardiac conduction system (CCS), mediates the propagation of this electrical impulse through the different cardiac compartments. The different components of the CCS are well described in mammals and distinguishable by anatomic, histologic and electrophysiological features (Davies et al, 1983; Massing and James, 1976; Schram et al., 2002; Viragh and Challice, 1977b). The sinoatrial node (SAN), localized in the right atrium, is responsible for the pacemaker function of the heart (Boyett et al., 2000). From this node, the impulse spreads through the atria and reaches the atrio-ventricular node (AVN). After a small delay, the impulse is then transferred to the ventricles through a specialized conductive system, which comprises the his bundle, the bundle branches (BB) and the Purkinje fibers (Pt). The gap junctions (GJs) ensure the electrical coupling between cardiomyocytes by connecting cytoplasms of two adjacent cells. The GJs are aggregates of intercellular channels formed by transmembrane proteins belonging to the connexin family (Cx) (Review, (Gros and Jongsma. 1996; van Rijen et al., 2001).
The structure and function of the CCS have extensively studied in big mammals (dog, rabbit, bovine) because this specialized tissue can be easily isolated from the compact layer of the heart (Davies et al. 1983). Nevertheless, the murine CCS is poorly characterized because of the impossibility to visualize these cells from the surrounding ventricular myocardium. As a result, there is no mouse CSS model available as of today.
However, there a great need for a mouse model since disturbances in the CCS leads to arrhythmias which may lead to sudden death as well as other cardiac medical conditions.
Few decades ago, it has been shown that the murine cardiac conductive cells can be recognized from the working myocytes by histological differences with electronic microscopy procedures (Viragh and Challice, 1 977a; Viragh and Challice, I 977b). However, this specialized tissue is undistinguishable from the ventricular wall when the ventricular cavities are exposed under a stereomicroscope. The detection of the CCS and electrophysiological measurements are not directly possible in mouse models stained for ACTH (Anumonwo et al., 2001) or in the CCS-LacZ transgenic mice expressing LacZ reporter gene in the developing cardiac conduction system (Rentschler et al., 2001). In both cases, the revelation of the CCS was done after fixation of the tissues that render impossible direct electrophysiological analyses.
Therefore, it is necessary to design alternative models that would allow such analysis. In this regard, we obtained transgenic mice expressing a reporter protein specifically in the CCS tissue which circumvent the above mentioned problems. This has been possible by targeting the connexin-40 (Cx40) locus.
In mammals, the connexin-40 (Cx40) is expressed in cardiomyocytes and vascular endothelial cells. In the heart, Cx40 is restricted to the atria and to the ventricular conduction system (AVN, His bundle, bundle branches and Purkinje fibers) and is not expressed in ventricular contractile myocytes (Coppen and Severs, 2002; Delorme et al., 1997; Delorme et al., 1995). In mice lacking the Cx40 gene, abnormal ECGs have been recorded and are associated with conduction defects in the right and left BB (Bevilacqua et al. 2000; Kirchhoff et al., 1998; Simon et al., 1998; Tamaddon et al., 2000; van Rijen et al., 2001).
We have generated transgenic mice in which the vital marker eGFP is expressed in the entire ventricular conduction system by knock-in the GFP gene at the Connexin 40 locus. eGFP is detected in the different components of the CCS such as the AVN, His bundle, bundle branches and Purkinje fibers. We have shown that eGFP cells present electrical features of conductive cardiomyocytes and that the anatomical description of the left and right bundle branches are correlated with their respective electrical activity maps recorded. These data give an accurate image of the entire mouse ventricular conduction system.
Our results show that the anatomical asymmetry observed between the right and left 1313 with GFP expression corresponds to a physiological reality as it is proven by the activation maps recorded for each branch. So, the propagation of the electrical activity follows the anatomic roads forming by the RBB and LBB revealed in GFP. These data confirm the hypothesis that the morphological discrepancy observed between the thin RBB and the large LBB may explain the occurrence of RBB block in Cx40 knockout mice while only slowing propagation was observed in the left branch (Tamaddon et al., 2000; van Rijen et al., 2001). It is noteworthy that in human patients a higher susceptibilty to develop RBB block than LBB block (Dorman et al., 2000).
Such a precise anatomic picture of the murine conductive tissue was never given before in the literature. These images of the murine CCS are identical to those given for the human heart a century ago, demonstrating a perfect conservation of this anatomic structure between the mouse and the big mammals (Tawara, 1906). We have shown that the morphology of the CCS fits perfectly with the propagation of the electrical activity in the heart.
So, we found that a structure function relationship exists between the GFP images and electrical activation maps.
The GFP is a powerful tool in molecular and cell biology (Hadjantonakis et al., 2002). The main advantage of using this reporter gene in our KI Cx40/GFP mouse model comes from the fact that this protein can he easily detectable on living tissues and cells. Therefore, the Cx40/GFP mice of the invention represent the first model in which this tissue is directly visualized on fresh tissue. This is a tremendous progress for performing electrophysiological studies of the mouse CCS as well as testing compounds which could be useful for preventing or treating various cardiac medical conditions.